Ciona Intestinalis and Styela Plicata (Tunicata)

Micron 69 (2015) 6–14

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Micron

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Ultrastructural aspects of naturally occurring wound in the tunic of

two ascidians: Ciona intestinalis and Styela plicata (Tunicata)

∗

Maria Antonietta Di Bella , Maria Carmela Carbone, Giacomo De Leo

Department of Biopatologia e Biotecnologie mediche e forensi, Sezione di Biologia e Genetica, Università degli Studi di Palermo, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Efﬁcient wound healing is essential for all animals from insects to mammals. Ciona intestinalis and

Received 10 July 2014

Styela plicata are solitary ascidians belonging to urochordates, a subphylum that occupies a key phy-

Received in revised form 10 October 2014

logenetic position as it includes the closest relative to vertebrates. Urochordate ﬁrst physical barrier

Accepted 27 October 2014

against invaders is the tunic, an extracellular matrix that is constantly exposed to all kinds of insults.

Available online 4 November 2014

Thus, when damage occurs, an innate immune response is triggered to eliminate impaired tissue and

potentially pathogenic microbes, and restore tissue functionality. Ultrastructural aspects of the tunic in

Keywords:

the wound healing process of two ascidians are described. In the injured areas, we evidenced thinning

Ascidians

Invertebrates of the tunic and areas of low ﬁbre density, dense intratunic bacterial and protozoan population, and

inﬂammatory aspects such as the increase in tunic cells, their aggregates, and phagocytosis. This is the

Wound healing

Ultrastructure ﬁrst report on tunic physical wounding occurring in the natural habitat.

1. Introduction consistency varies from gelatinous to leathery in the different

ascidian species, on account of variations in its constituents and the

In many organisms surface tissues are in permanent contact different arrangement of its ﬁbrils. Styela belongs to the suborder

with the environment and each animal has evolved repair mech- of Stolidobranchia and its tunic is leathery and hard, the ﬁbrils

anisms against physical injuries to restore tissue architecture and form regular bundles which are tightly interwoven and differently

homeostasis, and to protect the organism from opportunistic infec- oriented, whereas the tunic of C. intestinalis, that belongs to the

tion. suborder of Phlebobranchia, is a jelly-like coating and the ﬁbrils

Both the marine sessile species Styela plicata (Styelidae) and are arranged in a loose network (De Leo et al., 1981; Di Bella et al.,

Ciona intestinalis (Cionidae) belong to the subphylum tunicates 1998). The outermost ﬁbrous layer of the tunic, called the cuticle,

or urochordates that, together with cephalochordates and verte- is usually harder than the inner region; the latter is a thick layer

brates, are included in the phylum chordates (Delsuc et al., 2006). of cellulose ﬁbrils, proteins, and polysaccharides embedded in an

Their integumentary tissue intermediary between the interior and amorphous matrix that also harbours free cells and is called ground

exterior of the animal is the tunic which covers the whole body. substance or tunic matrix. Several types of free mesenchyme-like

The tunic is a multifunctional tissue that provides support and cells, known as tunic cells, varying from species to species, have

mechanical rigidity. In addition it is a surface barrier exposed to been described. They are involved in various tunic biological

constant microbial attack. It is unique tissue among metazoans, functions, such as accumulation of metabolites and inorganic

being an extracellular cellulosic matrix coating the epidermis, products, elimination of metabolic wastes, pigmentation, defence

whose components are proteins associated with carbohydrates response, phagocytosis, and allorecognition (Burighel and Cloney,

including cellulose or cellulose-like polysaccharides (De Leo et al., 1997 and references therein; Hirose, 2009a).

1977; Goodbody, 1974; Patricolo and De Leo, 1979). The tunic The cuticle can be frequently damaged by sands and microor-

ganisms and any rupture must be quickly repaired to prevent

microbes from gaining access and spreading throughout the body.

It frequently harbours foreign material including cyanobacteria

or algal symbionts attached to its external surface, even if host-

∗ associated symbionts can also be located within the tunic matrix

Corresponding author at: Dip. Biopatologia e Biotecnologie mediche e forensi,

of some ascidians (Blasiak et al., 2014; De Leo and Patricolo,

Università di Palermo, Via Divisi 83, 90133 Palermo, Italy. Tel.: +39 91 6554600;

1980; Hirose et al., 2006). Thus, when damage occurs, the tunic

fax: +39 91 6554624.

E-mail address: [email protected] (M.A. Di Bella). must possess repair mechanisms against physical wounding to

http://dx.doi.org/10.1016/j.micron.2014.10.006

M.A. Di Bella et al. / Micron 69 (2015) 6–14 7

Fig. 1. Electron micrographs of cross sections through the intact and damaged tunic from Styela and Ciona. Low power showing a general view of the Styela (A) and Ciona

(B) intact tunic: the undulating folds of the outer thin cuticular layer (cu) with foreign material encrusted on its external surface can be seen. The inset in (A) shows an area

of the same micrograph visualized at high magniﬁcation with microﬁbrils arranged in tightly packed bundles. (C) Section showing interruptions of Styela cuticle continuity

and loose ﬁne ﬁbrogranular material in the subcuticular area. (D–G) Ciona damaged cuticle. Note the breaks, the variable thickness, and loss of the density; outside the thin

cuticle debris and encrusting agents are present. (F) The micrograph illustrates how a group of foreign agents (bacteria, algae, etc.) is entrapped by inward cuticle folding,

and a point of cuticle discontinuity (inset) that allows the entry for pathogens to invade the subcuticular layer (F, G arrows); b = bacterium; gs = ground substance. Bars: A, B,

E, F, G = 5 ␮m; C, D = 2.5 ␮m; A, F insets = 0.5 ␮m.

re-establish normal architecture. As Ciona and Styela are basal with cells and an inﬂammatory response including encapsulation

invertebrate chordates lacking adaptative immune systems, tissue and often tissue damage occurs (Di Bella and De Leo, 2000;

damage triggers an innate immune response to protect from Parrinello and Patricolo, 1984; Parrinello et al., 1977, 1984);

infection. As observed during inﬂammatory-like response exper- soluble factors, and extracellular components can also collabo-

imentally induced, key players in these processes are tunic cells rate.

and their products, and haemocytes. In few hours after injec- The present ultrastructural study aims to describe aspects of

tion of soluble or particulate materials into the body wall of wound healing that occur in nature in the tunicates, S. plicata and

C. intestinalis, the tunic matrix appears to be densely populated C. intestinalis and to compare their cellular reactions.

8 M.A. Di Bella et al. / Micron 69 (2015) 6–14

Fig. 2. Tunic matrix in the main body of wounded Styela (A–E) and Ciona (F). Styela tunic ﬁbres do not form homogeneous tunic matrix and bundles are not so densely packed

and interwoven as usually observed. Conversely, branching ﬁbres are loose and form net-like aggregates or masses where tunic cells are entrapped or tightly adherent (C–E).

Some of these cells are seen while releasing ﬁbrillar material (C circle). (F) Ciona tunic matrix with loose, moderately dense ﬁbrillar network entrapping bacteria (b). Bars:

A = 5 ␮m; B, C = 0.5 ␮m; D, F = 2 ␮m; E = 2.5 ␮m.

2. Material and methods of the animals, the specimens were anaesthetized by adding MS222

to the medium (ﬁnal concentration 0.2%). Fragments from the tunic

C. intestinalis adult specimens about 3–5 cm long were obtained body 1–3 mm long were ﬁxed using the following procedure.

◦

from Stazione Zoologica Anton Dohrn, Naples, Italy. Adult speci- They were placed for 1 h at 4 C in a solution containing 1.5% glu-

mens of the ascidian S. plicata collected in the Ganzirri coast lake taraldehyde (Sigma–Aldrich, St. Louis, USA), buffered with 0.05 M

◦

(Southern Italy), were kept in aerated aquaria at 15–18 C until sodium cacodylate (pH 7.3 plus 1.7% sodium chloride), and post-

◦

used. Animals were fed daily with various food types including ﬁxed for 1 h at 4 C with 1% osmium tetroxide in 0.05 M sodium

freeze-dried rotifers, green unicellular algae and marine inverte- cacodylate at pH 7.3. All specimens were rinsed brieﬂy and dehy-

brate artiﬁcial diet coraliquid (Sera Heinsberg, Germany). drated in a graded series of ethanol solutions, cleared in propylene

Pieces of tunic cleaned by gentle brushing to remove debris and oxide and embedded in Epon resin.

fouling organisms were dissected from different regions of the body Semithin sections were stained with toluidine blue. Ultrathin

of apparently healthy specimens. To avoid extensive contractions sections (50–70 nm thick) were stained with uranyl acetate and

M.A. Di Bella et al. / Micron 69 (2015) 6–14 9

lead citrate and examined under a transmission electron micro-

scope (Philips CM 10), at 80 kV.

Negatives were scanned on an Epson Perfection V 700 PHOTO

scanner and acquired as TIFF ﬁles. All TIFF ﬁles were resampled

at 300 ppi and subsequently re-sized and optimized for brightness

and contrast by using Photoshop (Adobe Systems).

For tunic identiﬁcation, the nomenclature proposed by De Leo

(1992) was followed.

3. Results

We found some tunic areas from naïve S. plicata and C. intestinalis

where signs of inﬂammatory reaction were observable in semithin

sections. In order to conﬁrm these observations, we carried out an

ultrastructural analysis. The electron microscope showed regions

of damaged tunic where the architecture was different from that

usually observed. To provide the context for considering results,

we describe brieﬂy the basic morphology of intact tunic. As previ-

ously reported, the tunic consists of a rather thin outer cuticle and

a thick ground substance layer where cells are scattered. In both

Styela and Ciona, the cuticle is a highly dense structure sufﬁciently

compact to harder the surface of the tunic. It is composed of ﬁbro-

granular material that results in a homogenous closely interwoven

network (Fig. 1A and B). In S. plicata ground substance, elementary

ﬁbrils are densely packed and usually associated to form regular

bundles embedded in the amorphous matrix; the bundles are dif-

ferently oriented and interlaced with each other (Fig. 1A, inset).

The innermost zone of C. intestinalis tunic is a pale amorphous

layer consisting of a network of delicate ﬁbrils (Fig. 1B). Where

the cuticle was damaged, there was loss of cuticle continuity and

compactness, and the thickness varied so that the tunic matrix was

externally exposed. The subcuticular areas consisted of loose ﬁbril-

lar material and appeared not completely organized (Fig. 1C–E).

Entrapment of foreign organisms was seen (Fig. 1F and G). In the

Fig. 3. Micrographs of portions of Styela (A) and Ciona (B) wounded tunic. The num-

main body of C. intestinalis tunic matrix several areas of irregular ber of granulocytes is very high recalling a typical aspect of inﬂamed tissue. The

ﬁbre density were present (Fig. 2F). The S. plicata tunic ﬁbres did not cells mainly found in Styela tunic are vesicular granulocytes with a characteristic

amoeboid feature, and cytoplasm ﬁlled with numerous vesicles. Most cells found

form thick and interlaced bundles as seen in intact samples (Fig. 1A,

in Ciona are globular granulocytes. Several cells are degenerating in both Styela and

inset) but branching ribbons not homogeneously packed covered

Ciona samples (arrows) Bars: 5 ␮m.

the wound area, ﬁbrous materials were loosely intertwined. Large

tightly packed tunic masses were present and several cells were

seen entrapped in them (Fig. 2A–E).

The wounded areas appeared with the aspects of inﬂamed that formed net-like structures (Figs. 3 and 4F and I). The loss of

tissue. The distribution pattern of cells was different from the content and the conﬂuence of vacuoles gave rise to drastic conﬁgu-

unwounded samples as the cell number was highly increased rational changes so that the cells appeared swollen. The cytoplasm

beneath the cuticle and in the main tunic body both in Styela and devoid of organelles and the membrane presenting points of dis-

Ciona (Fig. 3A and B). continuity reduced the cells to ghosts (Fig. 4E).

Tunic clear vesicular granulocytes were the cell types most fre- In the case of C. intestinalis, tunic cells observed in these

quently found in Styela wounded tunic. They showed irregular wounded areas were conspicuously globular granulocytes whose

shape and amoeboid aspect as the cellular proﬁle exhibited vari- shape ranged from round to elongated (Fig. 3). Their cytoplasm was

ous protrusions and frequently petaloid features. Their cytoplasm entirely occupied by large globules containing highly dense mate-

was completely occupied by many vesicles both in the inner and rial and some vesicles in the residual cytoplasm. Conﬂuence of the

in the peripheral areas (Figs. 3A and 4A, G, L). Cells also showed vacuoles and ﬂaking of the electron-dense vacuolar material were

phagocytic features displaying irregular shape and cell surface observed (Fig. 5A and B). Some cells presented phago-lysosomes

often ruffled with filopodia. The cytoplasm often contained vesicles engulfing cellular debris and remnants of foreign or dead mat-

and phagosomes engulﬁng myelin-like membranes or other cells ter; moreover, some unilocular granulocytes characterized by the

(Figs. 3A and 4B and C). Heterogeneous material (bacteria, algae, cell presence of a single, large vacuole containing dark ﬁbrogranular

debris) was consistently observed both within intracellular spaces material were also found. They were observed going through pro-

in phagocytic vacuoles and free around the wound, within the tunic gressive modiﬁcations, as the large inclusion appeared uniformly

matrix (Figs. 3 and 4N). Some microgranulocytes showing several ﬁlled or losing the compactness and dissolving near the cell mem-

granules ﬁlled with more or less electron-dense material, globu- brane (Fig. 5C, D, E, and H). As observed in Styela, evidence of

lar granules or vacuoles with a meshwork of thin ﬁlaments inside advanced cell degranulation was found in these areas. Gather-

were also observed (Fig. 4D, M, H). Moreover, several cells were in ing of vesicles, cytoplasmic membrane dissolution and extrusion

a degranulating stage. The plasma membrane was interrupted and of the electron-dense contents released from the disintegrating

the cell content appeared released outside the cell. The discharged cell producing meshwork of thin ﬁbrils around were observed

material seemed to bind to the ﬁne ﬁlaments of the tunic matrix (Figs. 3 and 5F). Finally, one peculiar aspect observed at wound

10 M.A. Di Bella et al. / Micron 69 (2015) 6–14

Fig. 4. Styela tunic granulocytes observed in wounded areas. (A) Vesicular granulocyte with a characteristic amoeboid feature and the cytoplasm ﬁlled with numerous vesicles

(v). (B, C) Tunic phagocytic cells containing lysosomal vacuoles and large phagosomes (ph). (D) Tunic microgranulocyte. The nucleus (n) is eccentric and cisternae of RER can

be observed. Many granules are present in the cytoplasm, some of which are strongly electron-dense, and others are pale or dot-like. (E) Remnants of a globular granulocyte.

Interruption of the cytoplasm membrane can be observed (arrowhead). (F) Degranulating cell and detail of its surface (I). (G, L) Details of cytoplasmic peripheral vesicles.

(H) Enlargement of a vesicle free in the matrix and containing ﬁbrillar material. (M) Globules free in the matrix. (N) Bacteria scattered externally around the wound or being

engulfed in digestive vacuoles. Bars: A, B, D, F = 2 ␮m; E = 2.5 ␮m; C, M, N = 1.5 ␮m; G, I = 0.25 ␮m; H, L = 0.5 ␮m.

sites was the adhesive morphology of cells that showed extensive The most remarkable differences here reported between intact

membrane contact and seemed to form strong cellular clot (Fig. 6A). and wounded tunics, are:

Several cells adhering to each other were reduced to simple ghosts

as the organelles were lost, the globules and the inclusions were a) thinning of the tunic

dissolved, intracellular vesicles with debris within, and some large b) high presence of bacteria and protozoans within the tunic

vacuoles engulﬁng bacteria were often present implying phago- c) inﬂammatory aspects

cytic activities and clearance of invading microbes (Fig. 6B–D).

The tunic represents a ﬁrst line of tunicate defence against

pathogens and injury; naturally occurring damage may produce

4. Discussion disruption of the continuous cuticle layer with loss of its density

and thickness. Thinning of the cuticle and softening of the ground

Open wounds of the integuments represent an entry port for substance were found in diseased tunics from ascidian Halocynthia

bacteria and pathogens to invade the open circulatory system of roretzi with clinical signs of soft tunic syndrome. This infectious

tunicates and the inner tissues, so that tissue damage triggers an disease that often leads to mass mortality is due to a kinetoplastid

innate immune response involving the concerted action of both and causes a difference in the tunic hardness, and presence of areas

humoral and cell mediated responses to protect organisms and heal of low ﬁbre density and interlacement (Hirose et al., 2009c). Simi-

the wound. larly, in contrast with the intact specimens, the exposed Styela tunic

In the present study some ultrastructural aspects of wounded areas evidenced the absence of thick interlacing ﬁbres bundles, but

tunics of two ascidians S. plicata and C. intestinalis are reported. showed rather ﬁbrous material loosely packed or ribbon of ﬁbrils

M.A. Di Bella et al. / Micron 69 (2015) 6–14 11

Fig. 5. Ciona tunic granulocytes in the healing area. (A, B) Globular granulocytes with several globules occupying most of the cytoplasm. Note the irregular surface of the

cell in (B). (C–E) Unilocular granulocytes in different steps of ultrastructural changes. (G) Magniﬁcation of the framed area in (C), showing peripheral ﬁbrous complex. (H)

Enlargement of the cell inclusion in (E) showing its paracrystalline appearance. (F) Releasing of cellular ﬁbrogranular contents that seem to ﬂow forming a concentric network

around tunic matrix. m = mitochondria. Bars: B, C, E, F = 2 ␮m; A, D = 1.5 ␮m; G, H = 0.5 ␮m.

that form aggregates. Coarse net-like structures and branching (Di Bella and De Leo, 2000). It has also been shown that following

ﬁbres covered the wound surface in Ciona tunic and electron- injury, production of additional cells probably capable of rebuild-

dense ﬁbres were aggregating to form the boundary layer. Similar ing damaged tissue occurs in different ways such as proliferation of

features have been described in the cuticle restoration elicited by resident cell population or increased mitotic contribution (Di Bella

experimentally damaging operations in other ascidians such as and De Leo, 2010; Di Bella et al., 2005).

Aplidium and Botrylloides (Hirose et al., 1995, 1997a,b). Moreover, Active phagocytosis was evident within these areas. Many cells

the cuticular boundary formation with aggregates that become with cytoplasmic extensions similar to ﬁlopodia and phagocytic

gradually packed is functionally and structurally similar to the new vacuoles engulﬁng bacteria or remnants of dead cells were found.

wall formation seen in the allogeneic rejection reaction in Botryllus Granulocytes with their clearance properties are critical compo-

(Hirose et al., 1990; Tanaka and Watanabe, 1973) and allograft nents of innate immune response that involve speciﬁc mechanisms

rejection in S. plicata (Raftos, 1990). Thus aggregating ﬁbres could of recognition of pathogens to be eliminated. Previous studies have

induce the formation of new covering cuticle and tunic matrix. documented the presence of bacteria both inside the tunic and

The massive number of cells within the wounded areas in both on the cuticle of Ciona (De Leo and Patricolo, 1980; De Leo et al.,

Styela and Ciona may be due to the recruitment of haemocytes that 1981; Groepler and Schuett, 2003) and cyanobacteria have been

inﬁltrate from blood lacunae reaching the injured area. Although observed in the tunic from other ascidians (Hirose et al., 2006,

in this study the mode of occurrence of this inﬁltration is not clari- 2009b). Recently, Blasiak and co-workers (2014) have analysed the

ﬁed, haemocytes can pass through the mantle epithelium probably intratunic resident bacterial communities. Making a comparison

induced by humoral factors discharged from tunic cells, as reported with the exterior bacterial groups, the authors evidenced commu-

during the experimentally induced inﬂammatory-like response nity differences because some groups seem to be present only in the

12 M.A. Di Bella et al. / Micron 69 (2015) 6–14

Fig. 6. Ciona tunic granulocytes in the healing area. The cells in (A) and (B) are tightly adherent to each other with almost no space between them. The cluster in (B) consists

of cells completely swollen and changed in shape by formation of cytoplasmic extensions (C). Intracellular vesicles where bacteria are accumulated are shown (D). Bars:

4 ␮m.

exterior areas. In the wounded samples the observed high density at present, the ultrastructural observations in this study do not

of intratunical bacteria population and foreign microorganisms is enable us to distinguish between inﬁltrating cells and tunic cells

possibly due to a pathogenic infection. Although it is thought there by their cytomorphology. Nevertheless, it could be speculated that

might be a mutual association between bacteria and the ascidian tunic cells release chemiotactic factors that induce haemocytes

host, and the tunic bacterial community could protect the tunicate inﬁltration.

against fouling through the production of natural products (Kwan Inﬂammatory cells are not only responsible for clearing

et al., 2012), there is evidence that some microbial cells give rise to the wound site from pathogens and dead cells, but through

a defensive reaction. This implies a strong selection from ascid- degranulation they also respond to the recognition of foreign

ian species. The defence reaction, at least in part, is carried out agents and release enzymes and cytokine-like molecules. Indeed,

by the production of several antimicrobial peptides expressed by molecules recognized by antibodies against the mammalian pro-

tunic granulocytes. Antimicrobial peptides, ubiquitously found in inﬂammatory cytokines IL-1-a and TNF-␣, have been reported in

all kingdoms, are known to have rapid and efﬁcient effects against several ascidian species (Ballarin et al., 2001; Cima et al., 2004;

microbes (Bulet et al., 2004). It has been shown that several antimi- Parrinello et al., 2007; Raftos et al., 1991, 1992). Their presence

crobial peptides were released into the Ciona tunic matrix during increases during the rejection reaction which occurs when genet-

inﬂammatory-like response experimentally induced (Di Bella et al., ically incompatible colonies of Botryllus contact each other (Cima

2011) and in the naturally occurring injury sites (Di Bella et al., et al., 2006; Franchi et al., 2014) and also during inﬂammatory-like

2013). reaction challenged in the C. intestinalis body wall by inocula-

As regards Styela, bacteria and protozoans, usually, are not tion with LPS (Parrinello et al., 2007). As suggested by Menin

largely present within the tunic of healthy adults; thus, their high et al. (2005), cytokine-like molecules, analogously to vertebrate

presence in these damaged sites may be due to the bacterial infec- cytokine, are involved in the selective recruitment and activation

tion. of haemocytes in the sites of inﬂammation or in the wounded area.

Remodelling of tissue integrity is performed by immune cells As regards S. plicata, it has recently been shown that histamine,

present at the wound site; degranulating cells were consis- a biogenic amine playing a role in the regulation of vertebrate

tently observed and the electron-dense ﬁbrous material discharged inﬂammation and immunity, is stored inside granular haemocytes;

around them may denote the involvement of tunic cells to aid the it is promptly released after in vitro stimulation of haemocytes

regeneration of the tunic matrix and the cuticle. The discharged with different pathogen-associated molecules patterns (PAMPs).

contents probably rearrange the tunic matrix as observed in some The massive increment in the number of haemocytes present at

colonial and solitary ascidians both during allogeneic rejection the site of wounding after the injection of histamine into the tunic,

(Hirose et al., 1990; Tanaka, 1973; Tanaka and Watanabe, 1973) and is consistent with the role that this molecule may play in the reg-

during inﬂammatory-like response (De Leo et al., 1992); further- ulation of Styela immunity, involving the selective recruitment of

more, following experimentally induced injury, many of the cells effector cells into the tissues (de Barros et al., 2007; García-García

undergo drastic changes releasing their contents to become true et al., 2014). Moreover, the prophenoloxidase system is activated on

ghosts of cells (De Leo et al., 1996, 1997). Most of cells encountered the wound to produce melanization (Jackson et al., 1993; Johansson

in the wounded areas of Styela were clear vesicular granulocytes; and Söderhall, 1989). The prophenoloxidase system is a proteoly-

indeed, globular granulocytes showing an increase of irregular sur- tic enzyme cascade which recognizes minuscule amounts of cell

face were the predominant cells present in Ciona damaged tunic. wall products from microorganisms (lipopolysaccharide, peptido-

Clear vesicular granulocytes are amoeboid cells, with various forms glycan and glucans) and responds to the microbes by activation

and evidence of phagocytosis. They might be involved in cellular of the system and the subsequent generation of immune factors

defence mechanisms and scavenging functions. (Cerenius et al., 2008). Tunicate globular granulocytes such as those

As regards the involvement of tunic cell in the healing process, called elsewhere morula cells, on contact with foreign molecules,

the role of each type remains uncertain. Because some resident can degranulate and release the content of their vacuoles, mainly

tunic cells are similar in morphology to certain types of haemocytes, inactive prophenoloxidase that, once active, produces melanin

M.A. Di Bella et al. / Micron 69 (2015) 6–14 13

which accumulates in some areas of the tunic (Ballarin et al., Acknowledgments

2001; Cammarata and Parrinello, 2009; Cammarata et al., 2008).

Thus, different enzymatically catalysed cascades involved in clot- We thank the Stazione Zoologica Anton Dohrn (Naples-Italy) for

ting, melanization, and complement activation (Lavelle et al., 2010; providing animals. The authors are grateful to Dr. Sheila McIntyre

Maldonado-Contreras and McCormick, 2011) can recruit a variety for polishing the English. M. A. Di Bella and G. De Leo are sup-

of cell types (Arizza and Parrinello, 2009; Parrinello, 1996; Vizzini ported by the Italian Ministero della Istruzione, dell’Università e

et al., 2008) and induce the expression of characteristic innate della Ricerca (MIUR).

immune receptors (Dishaw et al., 2011; Parrinello, 2009; Sasaki

et al., 2009; Shida et al., 2003) and immunological phenomena

(Melillo et al., 2006; Smith and Peddie, 1992). References

Interestingly, in Ciona specimens, several tightly packed and

aggregating globular granulocytes were found at the site of injury. Arizza, V., Parrinello, N., 2009. Inﬂammatory hemocytes in Ciona intestinalis innate

immune response. Invertebr. Surviv. J. 6, S58–S66.

The exhibited tight clustering is morphologically similar to the for-

Ballarin, L., Franchini, A., Ottaviani, E., Sabbadin, A., 2001. Morula cells as the

mation of cellular clumps. A stable clump is required to seal the

main immunomodulatory haemocytes in ascidians: evidences from the colonial

wound and prevent the loss of haemolymph because no coagulation species Botryllus schlosseri. Biol. Bull. 201, 59–64.

Bulet, P., Stocklin, R., Menin, L., 2004. Anti-microbial peptides: from invertebrates

has been reported in the tunicate haemolymph and there is general

to vertebrates. Immunol. Rev. 198, 169–184.

agreement that extracellular clots do not occur (Jiang and Doolittle,

Burighel, P., Cloney, R.A., 1997. Urochordata: Ascidiacea. In: Harrison, F.W., Rup-

2003; Kulman et al., 2006). The aggregation of haemocytes also pert, E.E. (Eds.), Microscopical Anatomy of Invertebrates, vol. 15. Wiley-Liss, New

York, pp. 221–347.

acts as a secondary barrier to prevent bacterial and pathogenic

Blasiak, L.C., Zinder, S.H., Buckley, D.H., Hill, R.T., 2014. Bacterial diversity associated

dissemination from the injury site. In insects and horsecrab the

with the tunic of the model chordate Ciona intestinalis. ISME J. 8, 309–320.

sequestration of bacteria seems to occur within seconds (Cerenius Cammarata, M., Parrinello, N., 2009. The ascidian prophenoloxidase activating sys-

tem. Invertebr. Surviv. J. 6, 67–76.

and Söderhäll, 2011; Lesch et al., 2007; Theopold et al., 2014). The

Cammarata, M., Arizza, V., Cianciolo, C., Parrinello, D., Vazzana, M., Vizzini, A.,

possibility exists that lectins, functioning as cell-surface recep-

Salerno, G., Parrinello, N., 2008. The prophenoloxidase system is activated dur-

tors, acting as opsonins, can bind to the microorganism surface ing the tunic inﬂammatory reaction of Ciona intestinalis. Cell Tissue Res. 333,

481–492.

by one binding site and to the cell surface by another binding

Cerenius, L., Söderhäll, K., 2011. Coagulation in invertebrates. J. Innate Immun. 3,

site, thus forming a bridge between a phagocyte and bacteria. A

3–8.

large number of haemagglutinating lectins binding to a wide vari- Cerenius, L., Lee, B.L., Söderhäll, K., 2008. The proPO-system: pros and cons for its

ety of carbohydrates have been isolated from ascidians (Green role in invertebrate immunity. Trends Immunol. 29, 263–271.

Cima, F., Sabbadin, A., Ballarin, L., 2004. Cellular aspects of allorecognition in the

et al., 2006; Menin and Ballarin, 2008; Parrinello et al., 2007;

compound ascidian Botryllus schlosseri. Dev. Comp. Immunol. 28, 881–889.

Vasta and Marchalonis, 1983). Recently, a new member of the RBL

Cima, F., Sabbadin, A., Zaniolo, G., Ballarin, L., 2006. Colony speciﬁcity and chemio-

(rhamnose binding lectin) family was isolated from the colonial taxis in the compound ascidian Botryllus schlosseri. Comp. Biochem. Physiol. 145,

376–382.

ascidian Botryllus schlosseri. Its ability to agglutinate foreign parti-

de Barros, C.M., Andrade, L.R., Allodi, S., Viskov, C., Mourier, P.A., Cavalcante, M.C.,

cles was characterized and a multiple role in immunosurveillance

Straus, A.H., Takahashi, H.K., Pomin, V.H., Carvalho, V.F., Martins, M.A., Pavão,

and immunomodulation was hypothesized (Gasparini et al., 2008; M.S., 2007. The hemolymph of the ascidian Styela plicata (Chordata-Tunicata)

contains heparin inside basophil-like cells and a unique sulfated galactoglucan

Franchi et al., 2011). The presence in damaged areas of clumps

in the plasma. J. Biol. Chem. 282, 1615–1626.

of cellular ghosts with vesicles where bacteria are accumulated

De Leo, G., 1992. Ascidian hemocytes and their involvement in defence reactions.

implies that cells may be involved in the clearance of cellular Boll. Zool. 59, 195–213.

De Leo, G., Patricolo, E., 1980. Blue-green algalike cells associated with the tunic of

debris and invading microbes. Remarkably, when different Botryl-

Ciona intestinalis L. Cell Tissue Res. 212, 91–98.

lus colonies are brought into contact, a strong self–nonself reaction

De Leo, G., Patricolo, E., D’Ancona Lunetta, G., 1977. Studies on the ﬁbrous compo-

is provoked that results in cell clumping at the sites of contact nents of the test of Ciona intestinalis Linnaeus. I. Cellulose-like polysaccharide.

Acta Zool. (Stockh.) 58, 135–141.

(Oren et al., 2008; Saito et al., 1994). Cell aggregation as allogeneic

De Leo, G., Patricolo, E., Frittitta, G., 1981. Fine structure of the tunic of Ciona intesti-

response, was also reported in the colonial ascidian Aplidium (Ishii

nalis L. II. Tunic morphology, cell distribution and their functional importance.

et al., 2008). Acta Zool. (Stockh.) 62, 259–271.

In conclusion, the current work provides advances in our knowl- De Leo, G., Parrinello, N., Di Bella, M.A., 1992. Structural changes in granulocytes

involved in the lysis of the tunic during inﬂammatory-like reaction induced in

edge of S. plicata and C. intestinalis wound repair and outlines

Ciona intestinalis (Tunicata, Ascidiacea). Eur. Arch. Biol. 103, 113–119.

the changes that occur in their damaged tunic areas. Some of

De Leo, G., Parrinello, N., Parrinello, D., Cassarà, G., Di Bella, M.A., 1996. Encapsulation

the observed features recall the allogeneic rejection reactions and response of Ciona intestinalis (Ascidiacea) to intratunical erythrocyte injection.

I. The inner capsular architecture. J. Invertebr. Pathol. 67, 205–212.

the inﬂammatory-like reactions that occur in solitary and com-

De Leo, G., Parrinello, N., Parrinello, D., Cassarà, G., Russo, D., Di Bella, M.A., 1997.

pound ascidians. In order to maintain host–microbial interactions

Encapsulation response of Ciona intestinalis (Ascidiacea) to intratunical erythro-

after cuticle damage, mechanisms of innate response includ- cyte injection. II. The outermost inﬂamed area. J. Invertebr. Pathol. 69, 14–23.

Delsuc, F., Brinkmann, H., Chorrot, D., Hervé, P., 2006. Tunicate and not cephalochor-

ing barrier defenses, secretion of components like antibacterial

dates are the closest living relatives of vertebrates. Nature 439, 965–968.

peptides, recruitment of tunic cells and haemocyte inﬁltration,

Di Bella, M.A., De Leo, G., 2000. Hemocyte migration during inﬂammatory-like reac-

clumping, surely occur even if we are not sure which one occurs tion of Ciona intestinalis (Tunicata, Ascidiacea). J. Invertebr. Pathol. 76, 105–111.

ﬁrst. Di Bella, M.A., De Leo, G., 2010. Contribution of microscopy to the study of prolif-

erating blood cells in Ciona intestinalis immune response. In: Méndez-Vilas, A.,

Although we did not intended to provide a complete description

Díaz, J. (Eds.), Microscopy: Science, Technology, Applications and Education, vol.

of the events, since we could not observe the time course of these 1, pp. 111–115.

Di Bella, M.A., Cassarà, G., Russo, D., De Leo, G., 1998. Cellular components and tunic

processes with precision in living specimens, the study may con-

architecture of the solitary ascidian Styela canopus (Stolidobranchiata). Tissue

tribute to the debate on the evolution of innate immunity and help

Cell 30, 352–359.

to show how wound healing, tissue clearing, and the reconstruction Di Bella, M.A., Carbone, M.C., De Leo, G., 2005. Aspects of cell production in mantle

of damaged areas can occur. Wound repair is a multi-step process tissue of Ciona intestinalis L. (Tunicata, Ascidiacea). Micron 36, 477–481.

Di Bella, M.A., Fedders, H., Leippe, M., De Leo, G., 2011. Localization of antimicro-

that involves a complex network of signals and behaviours nec-

bial peptides in the tunic of Ciona intestinalis (Ascidacea, Tunicata) and their

essary to seal the wound; therefore, future investigations should

involvement in local inﬂammatory-like reactions. Results Immunol. 1, 70–75.

include the integration of morphological data with biochemical, Di Bella, M.A., Fedders, H., Leippe, M., De Leo, G., 2013. Antimicrobial peptides

in the tunic of Ciona intestinalis (Tunicata). In: Méndez-Vilas, A. (Ed.), World-

molecular and physiological results and research on the possible

wide Research Efforts in the Fighting Against Microbial Pathogens: From Basic

role of different genes simultaneously involved in the key immune

Research to Technological Developments. BrownWalker Press, Boca Raton, USA, defense mechanisms. pp. 63–67.

14 M.A. Di Bella et al. / Micron 69 (2015) 6–14

Dishaw, L.J., Giacomelli, S., Melillo, D., Zucchetti, I., Haire, R.N., Natale, L., Russo, structural and functional characterization of Ciona intestinalis C3a receptor. J.

N.A., De Santis, R., Litman, G.W., Pinto, M.R., 2011. A role for variable region- Immunol. 177, 4132–4140.

containing chitin-binding proteins (VCBPs) in host gut–bacteria interactions. Menin, A., Ballarin, L., 2008. Immunomodulatory molecules in the compound ascid-

Proc. Natl. Acad. Sci. U.S.A. 108, 16747–16752. ian Botryllus schlosseri: evidence from conditioned media. J. Invertebr. Pathol.

Franchi, N., Schiavon, F., Carletto, M., Gasparini, F., Bertoloni, G., Tosatto, S.C., Ballarin, 99, 275–280.

L., 2011. Immune roles of a rhamnose-binding lectin in the colonial ascidian Menin, A., del Favero, M., Cima, F., Ballarin, L., 2005. Release of phagocytosis-

Botryllus schlosseri. Immunobiology 216, 725–736. stimulating factor(s) by morula cells in a colonial ascidian. Mar. Biol. 148,

Franchi, N., Hirose, E., Ballarin, L., 2014. Cellular aspects of allorecognition in the 225–230.

compound ascidian Botrylloides simodiensis. Invertebr. Surviv. J. 11, 219–223. Oren, M., Escande, M.I., Paz, G., Fishelson, Z., Rinkevich, B., 2008. Urochordate

García-García, E., Gómez-González, N.E., Meseguer, J., García-Ayala, A., Mulero, V., histoincompatible interactions activate vertebrate-like coagulation system

2014. Histamine regulates the inﬂammatory response of the tunicate Styela components. PLoS ONE 3, e3123.

plicata. Dev. Comp. Immunol. 46, 382–391. Parrinello, N., 1996. Cytotoxic activity of tunicate hemocytes. In: Rinkevich, B.,

Gasparini, F., Franchi, N., Spola, B., Ballarin, L., 2008. Novel rhamnose-binding Müller, W.E.G. (Eds.), Invertebrate Immunology. Springer-Verlag, Berlin, pp.

lectins from the colonial ascidian Botryllus schlosseri. Dev. Comp. Immunol. 32, 190–217.

1177–1191. Parrinello, N., 2009. Focusing on Ciona intestinalis (Tunicata) innate immune system.

Goodbody, I., 1974. The physiology of ascidians. In: Russel, F.S., Yonge, M. (Eds.), Evolutionary implications. Invertebr. Surviv. J. 6, S46–S57.

Advances in Marine Biology, vol. 12. Academic Press, London, pp. 1–149. Parrinello, N., Patricolo, E., 1984. Inﬂammatory-like reaction in the tunic

Green, P., Luty, A., Nair, S., Radford, J., Raftos, D., 2006. A second form of collagenous of Ciona intestinalis (Tunicata), II. Capsule components. Biol. Bull. 167,

lectin from the tunicate, Styela plicata. Comp. Biochem. Physiol. 144, 343–350. 238–250.

Groepler, W., Schuett, C., 2003. Bacterial community in the tunic matrix of a colonial Parrinello, N., Patricolo, E., Canicattì, C., 1977. Tunicate immunobiology, I. Tunic

ascidian Diplosoma migrans. Helgoland Mar. Res. 57, 139–143. reaction of Ciona intestinalis L. to erythrocyte injection. Boll. Zool. 44,

Hirose, E., 2009a. Ascidian tunic cells: morphology and functional diversity of free 373–381.

cells outside the epidermis. Invertebr. Biol. 128, 83–96. Parrinello, N., Patricolo, E., Canicattì, C., 1984. Inﬂammatory-like reaction in the tunic

Hirose, E., Saito, Y., Watanabe, H., 1990. Allogeneic rejection induced by cut surface of Ciona intestinalis (Tunicata), I. Encapsulation and tissue injury. Biol. Bull. 167,

contact in the compound ascidian Botrylloides simodensis. Invertebr. Reprod. Dev. 229–237.

17, 159–164. Parrinello, N., Arizza, V., Cammarata, M., Giaramita, F.T., Pergolizzi, M., Vazzana, M.,

Hirose, E., Saito, Y., Watanabe, H., 1995. Regeneration of the tunic cuticle in the Vizzini, A., Parrinello, D., 2007. Inducible lectins with galectin properties and

compound ascidian, Botrylloides simodensis. Dev. Comp. Immunol. 19, 143–151. human IL1␣ epitopes opsonize yeast during the inﬂammatory response of the

Hirose, E., Saito, Y., Watanabe, H., 1997a. Subcuticular rejection: an advanced mode ascidian Ciona intestinalis. Cell Tissue Res. 329, 379–390.

of the allogeneic rejection in the compound ascidians Botrylloides simodensis and Patricolo, E., De Leo, G., 1979. Studies on the ﬁbrous components of the test of Ciona

B. fuscus. Biol. Bull. 192, 53–61. intestinalis Linnaeus, II. Collagen-elastin-like protein. Acta Zool. (Stockh.) 60,

Hirose, E., Taneda, Y., Ishii, T., 1997b. Two modes of cuticle formation in a colonial 259–269.

ascidian Aplidium yamazii. Dev. Comp. Immunol. 21, 25–34. Raftos, D., 1990. The morphology of allograft rejection in Styela plicata (Urochordata:

Hirose, E., Hirose, M., Neilan, B.A., 2006. Localization of symbiotic cyanobacteria in Ascidiacea). Cell Tissue Res. 261, 389–396.

the colonial ascidian Tridemnidum miniatum (Didemnidae; Ascidiacea). Zool. Sci. Raftos, D.A., Cooper, E.L., Habicht, G.S., Beck, G., 1991. Invertebrate cytokines: tuni-

23, 435–442. cate cell proliferation stimulated by an interleukin 1-like molecule. Proc. Natl.

Hirose, E., Neilan, B.A., Scmidt, E.W., Murakami, A., 2009b. Enigmatic life and evo- Acad. Sci. U.S.A. 88, 9518–9522.

lution of Prochloron and related cyanobacteria inhabiting colonial ascidians. In: Raftos, D.A., Cooper, E.L., Stillman, D.L., Habicht, G.S., Beck, G., 1992. Invertebrate

Gault, P.M., Marler, H.J. (Eds.), Handbook on Cyanobacteria. Bacteriology Devel- cytokines II: release of interleukin-1-like molecules from tunicate hemocytes

opments Series. Nova Science Publishers, Inc., pp. 161–189. stimulated with zymosan. Lymphokine Cytokine Res. 11, 235–240.

Hirose, E., Ohtake, S.-I., Azumi, K., 2009c. Morphological characterization of the tunic Saito, Y., Hirose, E., Watanabe, H., 1994. Allorecognition in compound ascidians. Int.

in the edible ascidian, Halocynthia roretzi (Drasche), with remarks on soft tunic J. Dev. Biol. 38, 237–247.

syndrome in aquaculture. J. Fish Dis. 32, 433–445. Sasaki, N., Ogasawara, M., Sekiguchi, T., Kusumoto, S., Satake, H., 2009. Toll-like

Ishii, T., Hirose, E., Taneda, Y., 2008. Tunic phagocytes are involved in allorejection receptors of the ascidian Ciona intestinalis: prototypes with hybrid function-

reaction in the colonial tunicate Aplidium yamazii (Polyclinidae, Ascidiacea). Biol. alities of vertebrate toll-like receptors. J. Biol. Chem. 284, 27336–27343.

Bull. 214, 145–152. Shida, K., Terajima, D., Uchino, R., Ikawa, S., Ikeda, M., Asano, K., Watanabe,

Jackson, A.D., Smith, V.J., Peddie, C.M., 1993. In vitro phenoloxidase activity in the T., Azumi, K., Nonaka, M., Satou, Y., Satoh, N., Satake, M., Kawazoe, Y.,

blood of Ciona intestinalis and other ascidians. Dev. Comp. Immunol. 17, 97–108. Kasuya, A., 2003. Hemocytes of Ciona intestinalis express multiple genes

Jiang, Y., Doolittle, R.F., 2003. The evolution of vertebrate blood coagulation as involved in innate immune host defense. Biochem. Biophys. Res. Commun. 302,

viewed from a comparison of puffer ﬁsh and sea squirt genomes. Proc. Natl. 207–218.

Acad. Sci. U.S.A. 100, 7527–7532. Smith, V.J., Peddie, C.M., 1992. Cell cooperation during host defence in the solitary

Johansson, M.W., Söderhall, K., 1989. Cellular immunity in crustaceans and the tunicate Ciona intestinalis (L). Biol. Bull. 183, 211–219.

proPO system. Parasitol. Today 5, 171–176. Tanaka, K., 1973. Allogeneic inhibition in a compound ascidian, Botryllus primigenus

Kwan, J.C., Donia, M.S., Hanb, A.W., Hirose, E., Haygood, M.G., Schmidt, E.W., 2012. Oka. II. Cellular and humoral responses in nonfusion reaction. Cell. Immunol. 7,

Genome streamlining and chemical defense in a coral reef symbiosis. Proc. Natl. 427–443.

Acad. Sci. U.S.A. 109, 20655–20660. Tanaka, K., Watanabe, H., 1973. Allogeneic inhibition in a compound ascidian,

Kulman, J.D., Harris, J.E., Nakazawa, N., Ogasawara, M., Satake, M., Davie, E.W., 2006. Botryllus primigenus Oka. I. Processes and features of “nonfusion” reaction. Cell.

Vitamin K-dependent proteins in Ciona intestinalis, a basal chordate lacking a Immunol. 7, 410–426.

blood coagulation cascade. Proc. Natl. Acad. Sci. U.S.A. 103, 15794–15799. Theopold, U., Krautz, R.S., Dushay, M., 2014. The Drosophila clotting system and its

Lavelle, E.C., Murphy, C., O’Neill, L.A., Creagh, E.M., 2010. The role of TLRs, NLRs, and messages for mammals. Dev. Comp. Immunol. 42, 42–46.

RLRs in mucosal innate immunity and homeostasis. Mucosal Immunol. 3, 17–28. Vasta, G.R., Marchalonis, J.J., 1983. In: Bog-Hanse, T.C. (Ed.), Lectins: Biol-

Lesch, C., Goto, A., Lindgren, M., Bidla, G., Dushay, M.S., Theopold, U., 2007. A role ogy, Biochemistry, Clinical Biochemistry, vol. 3. Walter De Gruyter, Berlin,

for hemolectin in coagulation and immunity in Drosophila melanogaster. Dev. pp. 461–468.

Comp. Immunol. 31, 1255–1263. Vizzini, A., Pergolizzi, M., Vazzana, M., Salerno, G., Di Sano, C., Macaluso, P., Arizza, V.,

Maldonado-Contreras, A.L., McCormick, B.A., 2011. Intestinal epithelial cells and Parrinello, D., Cammarata, M., Parrinello, N., 2008. FACIT collagen (1alpha-chain)

their role in innate mucosal immunity. Cell Tissue Res. 343, 5–12. is expressed by hemocytes and epidermis during the inﬂammatory response of

Melillo, D., Sfyroera, G., De Santis, R., Graziano, R., Marino, R., Lambris, J.D., Pinto, M.R., the ascidian Ciona intestinalis. Dev. Comp. Immunol. 32, 682–692.

2006. First identiﬁcation of a chemotactic receptor in an invertebrate species: